Biology subjects

Sigismondo, G.

Publications and source records attributed to Sigismondo, G..

4 recordsLinked to original sources

bioRxiv2025

Nucleotide insufficiency induced by p53 deficiency leads to replication stress driving genomic instability

P53 prevents DNA damage by inducing repair processes, cell cycle arrest or apoptosis. P53 loss leads to replication stress and genomic instability, yet the mechanisms underlying these effects and their contribution to catastrophic genomic events such as chromothripsis remain poorly understood. Using patient-derived fibroblasts with germline p53 variants, that spontaneously undergo chromothripsis, and p53-downregulated fibroblasts, we discovered that p53 loss leads to aberrant transcriptional upregulation, increasing nucleotide consumption while simultaneously decreasing nucleotide biosynthesis. This imbalance in production and consumption results in insufficient nucleotide pools, leading to replication stress and genomic instability, which are rescued by nucleoside supplementation or transcription normalization. The replication stress triggers telomere dysfunction, micronuclei formation, and ultimately chromothripsis. Emerging dominant chromothriptic clones exhibit normal DNA replication, telomere stabilization, and ecDNA, highlighting critical features for clonal selection. Hence, p53 coordinates transcription and nucleotide pools, crucial for maintaining genomic stability and preventing early cancer development.

cancer biology↗

bioRxiv2024

Isolation of proteins on chromatin (iPOC) reveals signaling pathway-dependent alterations in the DNA-bound proteome

Signaling pathways often convergence on transcription factors (TFs) and other DNA-binding proteins (DBPs) that regulate chromatin structure and gene expression, thereby governing a broad range of essential cellular functions. However, the repertoire of DBPs is incompletely understood even for the best-characterized pathways. Here, we optimized a strategy for the isolation of Proteins on Chromatin (iPOC) exploiting tagged nucleoside analogues to label the DNA and capture associated proteins, thus enabling the comprehensive, sensitive, and unbiased characterization of the DNA-bound proteome. We then applied iPOC to investigate chromatome changes upon perturbation of the cancer-relevant PI3K/AKT/mTOR pathway. Our results show distinct dynamics of the DNA-bound proteome upon selective inhibition of PI3K, AKT, or mTOR, and we provide evidence how this signaling cascade regulates the DNA-bound status of SUZ12, thereby modulating H3K27me3 levels. Collectively, iPOC is a powerful approach to study the composition of the DNA-bound proteome operating downstream of signaling cascades, thereby both expanding our knowledge of the mechanism of action of the pathway, and unveiling novel chromatin modulators that can potentially be targeted pharmacologically.

molecular biology↗

bioRxiv2021

Multi-layered chromatin proteomics identifies cell vulnerabilities in DNA repair

The DNA damage response (DDR) is essential to maintain genome stability, and its deregulation predisposes to carcinogenesis while encompassing attractive targets for cancer therapy. Chromatin governs the DDR via the concerted interplay among different layers, including DNA, histone post-translational modifications (hPTMs), and chromatin-associated proteins. Here we employ multi-layered proteomics to characterize chromatin-mediated functional interactions of repair proteins, signatures of hPTMs, and the DNA-bound proteome during DNA double-strand break (DSB) repair at high temporal resolution. Our data illuminate the dynamics of known and novel DDR-associated factors both at chromatin and at DSBs. We functionally attribute novel chromatin-associated proteins to repair by non-homologous end-joining (NHEJ), homologous recombination (HR) and DSB repair pathway choice. We reveal histone reader ATAD2, microtubule organizer TPX2 and histone methyltransferase G9A as regulators of HR and PARP-inhibitor sensitivity. Furthermore, we distinguish hPTMs that are globally induced by DNA damage from those specifically acquired at sites flanking DSBs ({gamma}H2AX foci-specific), and profiled their dynamics during the DDR. Integration of complementary chromatin layers implicates G9A-mediated monomethylation of H3K56 at DSBs in HR. Our data provide a dynamic chromatin-centered view of the DDR that can be further mined to identify novel mechanistic links and cell vulnerabilities in DSB repair.

cancer biology↗

bioRxiv2018

Spatiotemporal proteomics uncovers cathepsin-dependent host cell death during bacterial infection

Immune cells need to swiftly and effectively respond to invading pathogens. This response relies heavily on rapid protein synthesis and accurate cellular targeting to ensure pathogen destruction. In return, pathogens intercept this response to ensure their survival and proliferation. To gain insight into this dynamic interface, we combined click-chemistry with pulsed stable isotope labeling of amino acids (pSILAC-AHA) in cell culture to quantify the newly synthesised host proteome during macrophage infection with the model intracellular bacterial pathogen, Salmonella enterica Typhimurium (STm). We monitored newly synthesised proteins across different host cell compartments and infection stages, and used available proteomics data in response to lipopolysaccharide to deconvolute the STm-specific response. Within this rich resource, we detected aberrant trafficking of lysosomal proteases to the extracellular space and the nucleus, the latter of which correlated with signatures of cell death. Pharmacological cathepsin inhibition suppressed Caspase-11 dependent macrophage cell death, thus demonstrating an active role for cathepsins during STm induced pyroptosis. Our study illustrates that resolving host proteome dynamics during infection can drive the discovery of biological mechanisms at the host-microbe interface.

systems biology↗